Both platelet activation and thrombin-mediated clot formation are essential to hemostasis. However, perturbations in either of these two hemostatic mechanisms may result in the formation of pathogenic thrombi (blood clots) which block blood flow to dependent tissues. This is the case in a variety of life-threatening vascular diseases, such as myocardial infarction, stroke, peripheral arterial occlusion and other blood system thromboses. Since various biochemical pathways contribute to vascular disease, treatment and prevention may focus on either inhibiting platelets, inhibiting thrombin or directly dissolving the blood clot.
Therefore, strategies to control platelet aggregation and release are desirable in the treatment of vascular disease [L. A. Harker and M. Gent, "The Use of Agents that Modify Platelet Function in the Management of Thrombotic Disorders" in Hemostasis and Thrombosis, R. W. Colman et al., eds., pp. 1438-56, J. B. Lippincott, Co., Philadelphia, Penna. (1987)]. Furthermore, inhibition of platelet aggregation may also be desirable in the case of extracorporeal treatment of blood, such as in dialysis, cardiopulmonary bypass surgery, storage of platelets in platelet concentrates and following vascular surgery.
Inhibition of platelets is particularly complicated because many different mechanisms may cause activation. These mechanisms involve one of several different receptors on the platelet surface. Recent attention in this area has been directed to glycoprotein IIb/IIIa, the platelet fibrinogen receptor. This platelet surface protein self-associates as a two-chain complex in a calcium-dependent manner, upon stimulation of platelets with ADP, epinephrine, thrombin or prostaglandin derivatives and precursors thereof [S. J. Shattil et al., "Changes in the Platelet Membrane Glycoprotein IIb/IIIa Complex During Platelet Activation", J. Biol. Chem., 260, pp. 11107-14 (1985); G. A. Marguerie et al., "Human Platelets Possess an Inducible and Saturable Receptor Specific for Fibrinogen", J. Biol. Chem., 254, pp. 5357-63 (1979)]. This results in platelet aggregation mediated by a cross-linking between fibrinogen and the activated glycoprotein IIb/IIIa complexes of two platelets. Glycoprotein IIb/IIIa specifically binds to the Arg-Gly-Asp sequence present in fibrinogen [M. D. Pierschbacher and E. Ruoslahti, "Cell Attachment Activity of Fibronectin Can Be Duplicated By Small Synthetic Fragments of the Molecule", Nature, 309, pp. 30-33 (1984); K. M. Yamada and D. W. Kennedy, "Dualistic Nature of Adhesive Protein Function: Fibronectin and Its Biologically Active Peptide Fragments Can Autoinhibit Fibronectin Function", J. Cell Biol., 99, pp. 29-36 (1984); N. Ginsberg et al., "Inhibition of Fibronectin Binding to Platelets By Proteolytic Fragments and Synthetic Peptides Which Support Fibroblast Adhesion", J. Biol. Chem., 260, pp. 3931-36 (1985); E. F. Plow et al., "The Effect of Arg-Gly-Asp-Containing Peptides on Fibrinogen and Von Willebrand Factor Binding to Platelets", Proc. Nat. Acad. Sci. USA, 82, pp. 8057-61 (1985); T. K. Gartner and J. S. Bennett, "The Tetrapeptide Analogue of the Cell Attachment Site of Fibronectin Inhibits Platelet Aggregation and Fibrinogen Binding to Activated Platelets", J. Biol. Chem., 260, pp. 11891-94 (1985); M. Kloczewiak et al., "Localization of a Site Interacting With Human Platelet Receptor on Carboxy-Terminal Segment of Human Fibrinogen Gamma Chain", Biochim. Biophys. Res. Comm., 107, pp. 181-87 (1982)].
Specific inhibitors of glycoprotein IIb/IIIa, such as monoclonal antibodies [J. S. Bennett et al., "Inhibition of Fibrinogen Binding to Stimulated Human Platelets By a Monoclonal Antibody", Proc. Natl. Acad. Sci. USA, 80, pp. 2417-21 (1983); R. P. McEver et al., "Identification of Two Structurally and Functionally Distinct Sites on Human Platelet Membrane Glycoprotein IIb/IIIa Using Monoclonal Antibodies", J. Biol. Chem., 258, pp. 5269-75 (1983); B. S. Coller, "A New Murine Monoclonal Antibody Reports An Activation-Dependent Change in the Conformation and/or Microenvironment of the Platelet Glycoprotein IIb/IIIa Complex", J. Clin. Invest., 76, pp. 107-08 (1985)] and small Arg-Gly-Asp-containing peptides [T. K. Gartner and J. S. Bennett, supra], are less toxic, faster acting and have a shorter duration of effect as compared to aspirin, the most commonly used platelet inhibitor. Further, unlike aspirin, these compounds are effective against a number of different platelet aggregation mechanisms. Both Arg-Gly-Asp-containing peptides and antibodies toward glycoprotein IIb/IIIa demonstrate antithrombotic efficacy in in vivo models of thrombosis [Y. Cadroy et al., "Potent Antithrombotic Effects of Arg-Gly-Asp-Val (RGDV) Peptide In Vivo", Circulat., Part II, 75, p. II-313 (1988); B. S. Coller et al., "Antithrombotic Effect of a Monoclonal Antibody to the Platelet Glycoprotein IIb/IIIa Receptor in an Experimental Animal Model", Blood, 68, pp. 783-86 (1986); S. R. Hanson et al., "Effects of Monoclonal Antibodies Against the Platelet Glycoprotein IIb/IIIa Complex on Thrombosis and Hemostasis in the Baboon", J. Clin. Invest., 81, pp. 149-58 (1988); T. Yasuda et al., "Monoclonal Antibody Against the Platelet Glycoprotein (GP) IIb/IIIa Receptor Prevents Coronary Artery Reocclusion Following Reperfusing With Recombinant Tissue-type Plasminogen Activator in Dogs", J. Clin. Invest., 81, pp. 1284-91 (1988); B. S. Coller et al., "Inhibition of Human Platelet Function In Vivo With A Monoclonal Antibody", Annals Int. Med., 109, pp. 635-38 (1988)].
In order to effectively inhibit platelet aggregation, Arg-Gly-Asp-containing peptides must be administered at concentrations greater than 10.sup.-5 M. Such high dosages limit the commercial feasibility of those peptides. Monoclonal antibodies to glycoprotein IIb/IIIa are more potent inhibitors of platelet aggregation, but their synthesis in mouse hybridoma cells poses greater potential immunological complications [S. R. Hanson et al., supra]. In addition, Arg-Gly-Asp peptides and antibodies toward glycoprotein IIb/ IIIa fail to block platelet secretion. Therefore, these agents may have a limited effectiveness in vivo due to the proaggregating effects of released platelet elements and their subsequent cascade-like activation of the circulating platelet pool. Finally, monoclonal antibodies toward glycoprotein IIb/IIIa are known to induce thrombocytopenia in both sub-human primates and man [S. R. Hanson et al., supra; H. K. Gold et al., "Pharmacodynamic Study of F(ab').sub.2 Fragments of Murine Monoclonal Antibody 7E3 Directed Against Human Platelet Glycoprotein IIb/IIIa in Patients with Unstable Angina Pectoris", J. Clin. Invest., 86, pp. 651-59 (1990)].
Recent attempts to obtain more effective antiplatelet agents have centered around snake venoms, some of which contain glycoprotein IIb/IIIa inhibitors. These include the proteins carinatin, also known as "echistatin", purified from Echis carinatus [C. Ouyang et al., "Characterization of the Platelet Aggregation Inducer and Inhibitor from Echis carinatus Snake Venom", Biochim. Biophys. Acta, 841, pp. 1-7 (1985); European patent application no. 382,538]; trigramin, purified from Trimeresurus gramineus [T. F. Huang et al., "Trigramin", J. Biol. Chem., 262, pp. 16157-63 (1987); European patent application no. 317,053]; a novel homodimeric antiplatelet protein, "applaggin", isolated from the venom of Agkistrodon p. piscivorus [PCT application No. WO 90/08772]; and others "European patent application no. 382,451]. These glycoprotein IIb/IIIa inhibitors all belong to a family of related snake venom antiplatelet proteins referred to as "disintegrins". Another polypeptide antiplatelet agent, "decorsin", which is structurally related to the disintegrin family, has recently been isolated from the saliva of the leech Macrobdella decora [J. L. Seymour et al., "Decorsin", J. Biol. Chem. 265, pp. 10143-47 (1990)]. It is thus reasonable to conclude that many if not all blood feeding organisms contain an antiplatelet protein related to the disintegrin family.
All members of the disintegrin family contain a large number of cysteine residues, several intramolecular disulfide bonds and the sequence Arg-Gly-Asp. The Arg-Gly-Asp sequence in disintegrins is one possible interactive site for IIb/IIIa binding [B. Savage et al., "Binding of the Snake Venom-Derived Proteins Applaggin and Echistatin to the Arginine-Glycine-Aspartic Acid Recognition Site(s) on Platelet Glycoprotein IIbIIIa Complex Inhibits Receptor Function", J. Biol. Chem., 265, pp. 11766-72 (1990); M. S. Dennis et al., "Platelet Glycoprotein IIb-IIIa Protein Antagonist from Snake Venoms: Evidence for a Family of Platelet-Aggregation Inhibitors", Proc. Natl. Acad. Sci. USA, 87, pp. 2471-75 (1989)], although synthetic mutants of echistatin lacking the Arg residue still exhibit significant, though diminished, antiplatelet activity. Moreover, a disintegrin from Sistrurus m. barbouri contains a Lys-Gly-Asp for Arg-Gly-Asp substitution and still exhibits effective antiplatelet activity [R. M. Scarborough et al., "Characterization of a Potent and GpIIb-IIIa Specific Platelet Aggregation Inhibitor from the Venom of the Southeastern Pygmy Rattlesnake", Abstract, Circulation, 82, p. III-370 (1990)]. The disintegrins inhibit platelet aggregation by competitively inhibiting fibrinogen or von Willebrand factor binding to the glycoprotein IIb/IIIa receptor [Savage et al., supra]. Disintegrins have been found to indirectly inhibit platelet secretion and eicosanoid metabolism as a result of preventing close cell contact of platelets [B. H. Chao et al., "Agkistrodon piscivorus piscivorus Platelet Aggregation Inhibitor: A Potent Inhibitor of Platelet Activation", Proc. Natl. Acad. Sci. USA, 86, pp. 8050-54 (1989)].
Disintegrins have been evaluated as anti-thrombotic agents in models of acute platelet-dependent thrombosis and in models of thrombolysis of experimental thrombi [R. J. Shebuski et al., "Characterization and Platelet Inhibitory Activity of Bitstatin, A Potent Arginine-Glycine-Aspartic Acid-Containing Peptide from the Venom of the Viper Bitis arietans", J. Biol. Chem., 264, pp. 21550-56 (1989)]. These agents exhibit potent anti-thrombotic effects. However, as with the monoclonal anti-IIb/IIIa antibodies, administration of disintegrins is associated with a transient platelet thrombocytopenia in sub-human primates [S. R. Hanson et al., J. Clin. Invest., 81, pp. 149-58 (1988)] and thus, potentially in man.
Other approaches to the prevention and treatment of vascular disease is the antagonism of thrombin. Thrombin is both a mediator of clot formation and an agonist for platelet activation. Animal studies have shown that inhibition of thrombin alone is a highly effective mechanism for prevention of platelet thrombus formation [S. R. Hanson et al., "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-phenylalanyl-L-propyl-L-arginyl Chloromethyl Ketone", Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)]. Heparin, the most widely used thrombin inhibitor in treating vascular disease, does not inhibit thrombin directly. Therefore, it has limited efficacy in inhibiting platelets. This is because heparin activity is neutralized by platelet secretory components, e.g., platelet factor 4 [J. A. Jakubowski and J. M. Maraganore, "Inhibition of Coagulation and Thrombin-Induced Platelet Activities by a Synthetic Dodecapeptide Modeled on the Carboxy-Terminus of Hirudin, Blood, 75, pp. 399-406 (1990)].
An alternative to heparin is the direct thrombin inhibitor, hirudin, which binds to thrombin forming a stoichiometric complex [S. R. Stone et al., "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28 (1986)]. Recently, a novel class of hirudin-based peptides has been designed and characterized ]J. M. Maraganore et al., "Design and Characterization of Hirulogs: A Novel Class of Bivalent Peptide Inhibitors of Thrombin", Biochemistry, 29, pp. 7095-7101 (1990); copending U.S. patent application No. 549,388]. These peptides, called "Hirulogs", are bivalent inhibitors of thrombin, binding to both the catalytic and anion-binding exosite of the enzyme. Hirulogs have been shown to be effective inhibitors of arterial thrombosis in sub-human primates "A. Kelly et al., "Potent Antithrombotic Effects of a Novel Hybrid Antithrombin Peptide In Vivo", Abstract, Circulation, 82, p. III-603 (1990)] and to improve vessel patency in models of tPA-induced fibrinolysis [P. Klement et al., "Effects of Heparin and Hirulog on tPA-Induced Thrombolysis in a Rat Model", Abstract, Fibrinolysis, 4, p. 9 (1990)]. While hirulogs show promise for the treatment of arterial, platelet-dependent thrombosis, there will be many clinical circumstances where thrombin inhibition alone is insufficient to prevent thrombosis. This is due to the multiplicity of platelet activation agonists, whose importance as mediators of platelets activation may differ depending on the nature of thrombogenesis.
Despite the developments to date, the need still exists for a better inhibitor of platelet activation and thrombus formation. Such an agent should inhibit platelet activation in response to all physiological agonists without causing transient or long-lasting thrombocytopenia. At the same time, such a molecule should inhibit thrombin-mediated fibrin deposition at the site of a clot, thus preventing a clot from growing.